An internal master clock, which in mammals is located in a small area of the hypothalamus called the suprachiasmatic nucleus (SCN), coordinate a wide range of physiological process important for anticipating daily environmental fluctuations. The SCN is synchronized with the external environment primarily by light inputs from the retina. Though indirect evidence suggests that glutamate is the excitatory neurotransmitter providing this input, a definitive set of experiments to show this has thus far been impossible. Intrinsically photoreceptive retinal ganglion cells (ipRGCs) are a subset of cells in the retina that express melanopsin and project to the SCN where they package and release glutamate into synaptic vesicle using the vesicular glutamate transporter 2 (VGLUT2). Here, we will take advantage of these two distinct features of ipRGCs (melanopsin and VGLUT2 expression) to create an otherwise fully functional retinal and circadian system that lacks only one feature: the ability to use glutamatergic transmission. Mice in which lox-P sites are inserted into the VGLUT2 gene flanking the second axon will be mated with mice expressing Cre-recombinase exclusively under the control of the melanopsin promoter (Opn4). Thus, the Cre-lox recombination-resulting in genetic deletion of VGLUT2-will take place only in ipRGCs. A number of possible co-transmitters, most notably pituitary adenylate cyclase activating peptide (PACAP), have been suspected to also play a role in mediating the light inputs and this model allows us to test whether these can mediate the effects of light on the circadian system in the absence of glutamate. Fundamental properties of the effects of light on the circadian timing system will be examined in VGLUT2 deleted and control mice: entrainment to 12 hour light-dark cycles, phase shift magnitudes in response to light pulses at varying intensities, entrainment to a 6-hour phase shift, light masking and constant lighting conditions. PUBLIC HEALTH RELEVANCE: Timing of physiological processes such as hormone release, body temperature, blood pressure in anticipation of daily environmental cycles is governed by an internal master clock, which in mammals is located in the suprachiasmatic nucleus (SCN) of the brain. Light keeps the master clock synchronized with the environment and here we will examine how the retina signals this stimulus to the SCN. Societal demands such as shift work can make it difficult for humans to receive these synchronizing inputs and this can impact arousal and wakefulness;moreover certain disorders such as seasonal affective disorders are also linked to light inputs to the master clock.